Dilute working stocks of libraries and double-stranding primers to 10uM

Dilute working stocks of sequencing primers to 3.2uM (6.4uL of stock solution in 193.6uL water)

Some considerations:

Oligos should be the maximum length because this will help with PCR cleanup and ligation efficiency

Make sure you have some spacer sequence around the restriction site. NEB has a list of the length of the spacer sequence required for each restriction enzyme. (8bp is usually a safe bet)

Order the lowest concentration allowable for the size oligo you want – this will be 50nmole for the 100bp oligo. This will already be more than you’ll need.

If you don’t mind spending more money you can order special “doped” oligo pools where instead of even concentrations of A/T or A/T/C/G or A/T/C, you get 90%A/2%C/8%G, etc. This allows for you to generate a library which is much more likely to produce productive clones.

Double strand the library with modified PCR

Expected max library size is 108 molecules (limit set by transformation efficiency.) You want to load 10X the expected library size for a single library construction. Therefore, you would like to have 109 molecules for a single transformation.

Perform PCR cleanup on the double-stranded library

This concentrates the samples and allows for the buffer to be switched to something more appropriate.

PCR purification columns can handle up to 10ug of DNA

100pmol of a 100bp oligo is about 3ug, so multiple 100-ul reactions of 25pmol can be combined into one column

Expected recovery from a PCR purification reaction is 90% (from the Invitrogen package)

You can run a sample of the PCR product out on a gel against a sample of the original library to verify that the double stranding worked (double stranded DNA should run slightly faster than single stranded)

Three libraries ~100bp; on the left is the single-stranded oligo; on the right are double-stranded oligos (different lanes are different primers)